Currently used techniques for detection of ligands are based on recognition of the protein by an antibody (ELISA, FACS) or by cells (bioassay), the latter offering the advantage of measuring only bioactive protein. However, increasing sensitivity is required to obtain a more complete image of biological systems and pathological processes. For instance, an immune answer can be characterized by a Th1 response (IL-2, IL-12, TNF and IFNy secretion) or Th2 response (IL-4, IL-5, IL-6 and IL-10 secretion) from which the cytokines are usually present in very low concentrations in primary cell cultures.

Amplification systems, enhancing the signal output generated by the receptor-ligand binding have been described, amongst others in W00018962 and W09964862. Those methods are, however, in vitro-based methods that may detect biological irrelevant interactions.

The present invention combines the advantages of the bioassay to a very high sensitivity, by inserting an amplification loop between the signal, induced by the binding of the ligand to be detected to its receptor, and the reporter system. The amplification loop essentially consists of a recombinant gene comprising a promoter, inducible by the signaling pathway of at least one of said receptors, whereby said signaling pathway is activated by binding of a specific ligand (called ligand A) to this receptor and whereby said promoter is operationally linked to a coding sequence encoding ligand A. Binding of the ligand to be detected activates the amplification loop, which on its turn activates the reporter system.

Alternatively, the amplification loop essentially consists of a recombinant gene comprising a promoter, inducible by the signaling pathway of a constitutively activated receptor, whereby said promoter is operationally linked to a coding sequence encoding said constitutively activated receptor.

It is a first aspect of the invention to provide an eukaryotic cell comprising a) one or more receptors binding a ligand A b) a recombinant gene comprising a promoter, inducible by the signaling pathway of at least one of said receptors, whereby said

signaling pathway is activated by binding of ligand A to this receptor and whereby said promoter is operationally linked to a coding sequence encoding ligand A c) a reporter system, inducible by binding of ligand A to at least one of said receptors. A reporter system can be any system that allows the detection and/or the selection of the cells carrying a receptor, binding ligand A, according to the invention. It is clear for the person skilled in the art that several reporter systems can be used. As a non-limiting example, a luciferase gene, an antibiotic resistance gene or a cell surface marker gene can be placed after a promoter that is induced by the signaling pathway. In such case, the coding sequence of the reporter gene is preferentially operationally linked to a promoter that is induced upon binding of ligand A to at least one of the receptors.

However, said promoter may be induced in an indirect way too, e. g. by binding of a ligand B to its receptor, whereby the coding sequence of ligand B is operationally linked to a promoter inducible by binding of ligand A to at least one of the receptors (ligand B being a specific ligand, different from ligand A). Alternatively, reporter systems may be used that are based on the change in characteristics of compounds of the signaling pathway, when said pathway is active, such as the phosphorylation and/or dimerisation of such compounds.

The receptors used may be any naturally occurring receptor or chimeric receptor, including, but not limited to nuclear receptors such as steroid receptors, G-coupled receptors and multimerizing receptors, such as cytokine receptors. The use of a chimeric receptor has the advantage that the amplification loop may be initiated by the interaction of a ligand B to a chimeric receptor, comprising a ligand B binding domain and a signaling domain of the receptor for ligand A. A preferred embodiment is an eukaryotic cell according to the invention whereby said receptor is a multimerizing receptor, preferably a cytokine receptor.

As mentioned above, the amplification loop may be initiated by any extracellular or intracellular event that can induce the promoter inducible by binding of ligand A to at least one of the receptors, such as by the induction of the expression of a constitutively activated receptor activating said promoter, or by binding of a ligand B to a receptor, preferably a chimeric receptor, whereby said promoter is activated.

The eukaryotic cell according to the invention may be any eukaryotic cell. Preferably, said eukaryotic cell is a mammalian cell, more preferably it is a HEK293 cell.

Another aspect of the invention is an eukaryotic cell according to the invention, whereby the coding sequence encoding ligand A, and operationally linked to a

promoter inducible by binding of ligand A to at least one of the receptors is placed downstream or upstream of an IRES sequence. Indeed, the amplification loop may switch on spontaneously, e. g. by promoter leakage, resulting in ligand A production and amplification. To avoid this, a sequence encoding a conditional lethal gene product is operationally linked to a promoter that is induced upon binding of ligand A to at least one of the receptors, whereby said sequence is followed by an IRES sequence operationally linked to a coding sequence encoding ligand A. Alternatively, ligand A is operationally linked to a promoter that is induced upon binding of ligand A to at least one of the receptors, whereby said sequence is followed by an IRES sequence operationally linked to a sequence encoding a conditional lethal gene product. Under conditions where no ligand A production is wanted, the product of the conditional lethal gene product is activated, resulting in the death of the cell with a leaking promoter.

Under conditions where ligand A production is wanted, the product of the conditional lethal gene is inactivated, and ligand A is translated from the IRES sequence.

Activation and/or inactivation of the conditional lethal gene may be an active proces, e. g. by modification of the conditional lethal gene product. Preferably the activation or inactivation is switched on by adding a compound to the medium. Alternatively, it may be an activity that can only exert its toxic effect in the presence of another compound.

As a non limiting example, thymidine kinase of the Herpes Simplex Virus (TK-HSV) can be used as conditional lethal gene; this is toxic in presence of gancyclovir and harmless when no gancyclovir is present in the medium.

Another aspect of the invention is a eukaryotic cell according to the invention whereby at least one of the receptors, binding ligand A, or a part of this receptor is encoded by an inducible gene. As a non-limiting example, a part of the receptor, as used here, may be one chain of a heteromerising receptor (illustrated in figure 1). By making the receptor inducible, autoinduction of the amplification loop can also be avoided: when the receptor is not expressed, leakage of the promoter and ligand A synthesis will not result in an amplification of ligand A. Under conditions where ligand A synthesis is wanted, the gene encoding the receptor is switched on, resulting in a functional receptor binding ligand A, and an induction of the amplification loop in presence of ligand A. Moreover, by the use of an inducible receptor, the amplification loop can be switched off if needed. Preferably the inducible receptor is induced by adding a compound to the medium.

Still another aspect of the invention is the use of a eukaryotic cell according to the invention to detect a ligand and/or to measure the concentration of said ligand. Said ligand may be, but is not necessarily ligand A. Indeed, as mentioned above, by the use of a chimeric receptor, the amplification loop may be switched on by binding of ligand B to the ligand binding domain of the chimeric receptor, whereby said receptor is inducing a promoter that is normally switched on by binding of ligand A to its receptor.

One preferred embodiment is the use, according to the invention, whereby said ligand is ligand A. Another preferred embodiment is the use, according to the invention whereby said ligand is a cytokine.

Definitions Receptor as used here does not necessarily indicate a single polypeptide, but may indicate a receptor complex, consisting of two or more polypeptides. Recombinant receptor means that at least one of said polypeptides is recombinant. A chimeric receptor is a recombinant receptor whereby the recombinant polypeptide comprises domains that are derived from at least two receptors.

Polypeptide as used here means any proteineous structure, independent of the length and includes molecules such as peptides, phosphorylated proteins and glycosylated proteins. Polypeptide as used herein is not necessarily indicating an independent compound but can also be used to indicate a part of a bigger compound, such as a domain of a protein.

Ligand means every compound that can bind to the ligand-binding domain of a receptor and that is able to initiate the signaling pathway by binding to said ligand binding domain, or every compound that can mimic this effect. Ligand A and ligand B can be any ligand ; however, in one experimental set up all ligands A are identical to each other, and all ligands B are identical to each other, but ligand A is different from ligand B.

Signaling pathway as used here does not imply that other compounds than ligand and receptor are involved: the signaling may be caused by translocation of the receptor, which directly induces the transcription of the target genes. Initiating as used here means starting the events that normally directly follow the binding of the ligand to the ligand-binding domain of a receptor, such as, as a non-limiting example, multimerization for a multimerizing receptor.

Compound means any chemical or biological compound, including simple or complex organic or inorganic molecules, peptides, peptido-mimetics, proteins, antibodies, carbohydrates, nucleic acids or derivatives thereof.

Bind (ing) means any interaction, be it direct or indirect. A direct interaction implies a contact between the binding partners. An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two compounds.

This interaction can be completely indirect, with the help of one or more bridging compounds, or partly indirect, where there is still a direct contact that is stabilized by the interaction of one or more compounds.

Operationally linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A promoter sequence"operationally linked"to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the promoter sequence.

An IRES sequence operationally linked to a coding sequence means that ribosome entry and initiation of translation for said coding sequence can start from the IRES sequence.

Brief description of the figures Figure 1: Principle of the amplification loop, illustrated by the doxycyclin-induced cytokine cascade. Cells stabile carrying the TRE2-IFNaR1/gp130 construct will only produce IFNaR1/gp130 after stimulation of the TRE2 promoter with doxycyclin, a process that acts via activation and inhibition of a doxycyclin-dependent transcriptional activator and repressor, respectively (not shown in the figure). IFNaR1/gp130 associates with the endogenous IFNaR2 to form a functional receptor complex, capable of activating the rPAP promoter after IFNp stimulation. Weak induction of the rPAP promoter (for instance via a chimeric receptor) in the absence of doxycyclin will not result in cell survival in puromycin-containing medium. After doxycyclin treatment, a loop is formed that enables FN (5 to gradually increase its own production, resulting in a puromycin resistance of the cell.

Figure 2: A. Overview of vector cloning. Vectors from which fragments were transferred to the backbone of other vectors by means of PCR or restriction fragment

exchange, are shown at the left of the converging arrows. B. Schematic representation of the final constructs pUTrPAP-TK-IRES-IFNß or pUTrPAP-TK-IRES-LIF.

Figure 3 : Triggering of the LIF (Leukemia Inhibiting Factor) cytokine cascade by a LIF concentration series after co-transfection of the cells with pXP2rPAP-luci. Cells were transfected with pUTrPAP-TK-IRES-LIF (gray bars) or pUTrPAP-TK-IRES-IFNß (black bars) and in both cases co-transfected with pXP2rPAP-luci. The cells were subsequently trypsinized and seeded in 24-well microtiterplates, stimulated with a serial dilution of LIF as indicated and tested for luciferase activity.

Figure 4: Triggering of the LIF cytokine cascade by a LIF concentration series in HEKrPAP-luci cells. Cells, stably transfected with pXP2rPAP-luci (HEKrPAP-luci) were transfected with pUTrPAP-TK-IRES-LIF (gray bars) or pUTrPAP-TK-IRES-IFNß (black bars). The cells were subsequently trypsinized and seeded in 24-well microtiterplates, stimulated with a serial dilution as indicated of LIF and tested for luciferase activity.

Figure 5: Triggering of the LIF cytokine cascade by limited LIF incubation times after co-transfection of the cells with pXP2rPAP-luci. Cells were transfected with pUTrPAP- TK-IRES-LIF (gray bars) or pUTrPAP-TK-IRES-IFNß (black bars) and in both cases co-transfected with pXP2rPAP-luci. The cells were subsequently trypsinized and seeded in 24-well microtiterplates, stimulated with a fixed LIF concentration (1 ng/mi) during different time periods (full bars) or left without LIF (hatched bars), washed, and finally tested for luciferase activity.

Examples Materials and methods to the examples For development of stabile cell lines, the plasmids pUTrPAP-TK-IRES-LIF and pUTrPAP-TK-IRES-IFNß were constructed. For this reason the constructs have inserted the TK (HSV) gene between the rPAP promoter and the cytokine cDNA sequence, allowing for selection of stabile transfected clones with a strictly regulated rPAP promoter. These plasmids however, will in first instance be used in transient transfection experiments for demonstration of the principle of a cytokine amplification loop.

The construction of these vectors is schematically shown in figure 2.

Construction of pUT-rPAP-TK Construction of pXP2-rPAP-TK First, the luciferase gene from the reporter construct pXP2rPAP-luci (Lavens D., licentiate thesis) was replaced by the gene for blasticidin resistance. For this, A PCR fragment containing the cDNA coding for blasticidin resistance was made using oligo's MBU-0-721 and MBU-0-722 and using pcDNA6/V5-HisA (Invitrogen) as a template.

As such, an Xhol site at the 5'end and an Xbal site at the 3'end of the gene was attached. After ligation in pCR-blunt, the resulting plasmid was digested with Xhol and Xbal and ligated in the Xhol-Xbal opened pXP2rPAP-luci vector such that the blasticidin resistance gene was operationally linked to the rPAP promoter. The plasmid was named XP2-rPAP-blasti. After this, the gene for blasticidin resistance was replaced by the TK (HSV) cDNA. For this, a PCR fragment containing the gene for TK (HSV) was made using oligo's MBU-0-976 and MBU-0-977 and plasmid pPHT-Hyg (BCCM, Belgium) as a template, thereby creating an EcoRl site at the 5'end and Xhol and Notl sites at the 3'end of the gene. After ligation in pCR-blunt, the resulting plasmid was digested with EcoRl and Xhol and ligated in the EcoRI-Xhol opened pXP2rPAP-blasti vector such that the TK (HSV) gene was operationally linked to the rPAP promoter. The resulting construct was named pXP2-rPAP-TK.

Construction of pUT6-16TK The 6-16 promoter was digested from the plasmid p6-161uci (kindly provided by Dr. S.

Pellegrini, Institut Pasteur, Paris) by a Hindi ! ! digest, blunted by Klenow polymerase and ligated into the pCR-blunt vector. A fragment containing the 6-16 promoter was isolated by a Spel and EcoRV digest and inserted upstream the gene for ß-galactosidase in the Spel-Pmll opened pUT-651 vector (Eurogentec, Seraing), resulting in the plasmid pUT6-16-ßgal. The gene for ß-galactosidase was then replaced by the TK cDNA sequence as follows : the plasmid pPHT-Hygro (BCCM, Belgium) was digested with Hindlll, blunted with Klenow polymerase, digested with Hindll and ligated in pCR-blunt. This plasmid was digested with Spel, blunted with Klenow, digested with Notl and ligated in the Hindll-Notl opened pUT6-16-ßgal plasmid, resulting in the plasmid pUT6-16-TK.

Construction of pUT-rPAP-TK From the pXP2rPAP-TK plasmid, a fragment containing the rPAP promoter and a short 5'end of the TK cDNA sequence was isolated by digestion with Nhel, blunting the end with Klenow polymerase, and a subsequent digest with BsiWI. This fragment was then transferred to the Spel (also blunted with Klenow polymerase)-BsiWI opened pUT6-16- TK vector such that the gene for TK (HSV) was operationally linked to the rPAP promoter in the pUT backbone. The resulting plasmid was named pUTrPAP-TK.

Construction of rPAP-TK-IRES-puro . Creation of Ncol site in IRES sequence We planned to insert an IRES sequence, containing an Ncol site at its start codon, downstream the TK gene in pUTrPAP-TK, allowing for production of polycistronic TK-cytokine messengers whereby the cytokine sequences could easily be exchanged.

To accomplish this, we first introduced an Ncol restriction site (mutagenesis of CCATGA-> CCATGG) at the start codon of the IRES sequence in the pIRESpuro2 vector (Clontech). The mutagenesis was performed with the"QuickChange"site- directed mutagenesis kit (Stratagene), according to the manufacturers guidelines, making use of oligonucleotides MBU-0-967 and MBU-0-968.

Construction of pUTrPAP-TK-IRES-puro A PCR fragment containing the IRES sequence and the gene for puromycin resistance, containing an Ncol site at its start codon, was made using oligo's MBU-0-1032 and MBU-0-981, using the mutated pIRES-puro2 as a template. As such, an EcoRV (blunt) site and a Notl site was added at the 5'and 3'end of the fragment, respectively. This fragment was ligated in pCR-blunt, resulting in the plasmid pCR-blunt-IRES-puro. The latter plasmid was then digested with EcoRV and Notl to create a fragment that contained the IRES sequence and the gene for puromycin resistance. The fragment was then inserted in the pUTrPAP-TK vector, which was first opened with Tth111 (located downstream the TK gene), blunted with Klenow polymerase, and subsequently digested with Notl. As such, a blunt end-Notl ligation resulted in a plasmid that contained in 5'to 3'direction: the rPAP promoter, the TK gene, the IRES sequence and the gene for puromycin resistance with an Ncol site at its start codon (pUTrPAP-TK-IRES-puro).

. Construction of rPAP-TK-IRES-LIF and rPAP-TK-IRES-IFN Because of the Ncol site at the start codon and the Xbal site at the 3'end of the puromycin resistance gene, it now becomes possible to quickly insert other genes downstream the TK-IRES sequence. Hence, since we first wanted to introduce the cDNA of LIF (Leukemia Inhibiting Factor) and IFNß (interferon ß) downstream the TK gene, these two sequences were produced by PCR using forward oligonucleotides that created an Ncol site at their start codon and reverse oligonucleotides that added an Xbal site at the 3'untranslated end. For these reactions, the forward oligonucleotides MBU-0-1016 (LIF) and MBU-0-1018 (IFNß) and reverse oligonucleotides MBU-O- 1017 (LIF) and MBU-0-1019 (IFNß) were used. The reactions were performed on cDNA of PMA-stimulated K562 cells for LlF and on the plasmid pATHIFNpg41 (BCCM, Belgium) for IFNß. The fragments were cloned in the pCR-blunt vector and named pCR-blunt-LIF and pCR-blunt-lFNß, respectively.

From these vectors, respectively LIF and IFNß cDNA containing fragments were isolated by an Ncol-Xbal digest. The fragments were then ligated in an Ncol-Xbal opened pUTrPAP-TK-IRES-puro vector that was dephosphorylated by calf intestinal alkaline phosphatase (CIAP ; Invitrogen-Life Sciences) because frequently problems arose as a result of intramolecular ligation of this vector. As such, the puromycin gene was exchanged by the cytokine cDNA sequences which were operationally linked to the rPAP-TK-IRES sequence. The corresponding plasmids were named pUTrPAP- TK-IRES-LIF and pUTrPAP-TK-IRES-IFNß.

Table 1: Oligonucleotides used in cloning and subcloning steps, leading to plasmids pUTrPAP-TK-IRES-LIF and pUTrPAP-TK-IRES-IFNß

Development of HEK rPAP-luci cells Hek 293-Flp-In cells (Invitrogen) were transfected with pM5neo-mEcoR (kindly provided by Dr. S. Kinoshita, Stanford University, Stanford) according to the calciumphosphate DNA-precipitation technique. This plasmid contained the gene for the ecotropic receptor for moloney murine leukemia virus. The cells were selected in 500pg/ml G418. The pool of G418 resistant cells was co-transfected with the plasmids pXP2rPAP-luci and plRES-puro2 (Clontech) at a ratio of 4: 1, again according to the calciumphosphate DNA-precipitation technique. Puromycin selection was applied at 1 pg/ml and single colonies were isolated which were individually tested for responsiveness to LIF (1 ng/ml). The strongest inducible clone with a maximal signal/noise ratio was selected and named HEKrPAP-luci.

Example 1 : Co-transfection with pXP2rPAP-luci and treatment of cells with a LIF concentration gracient.

To demonstrate the principle of the cytokine cascade in a transient transfection experiment, HEK293 cells were transfected with pUTrPAP-TK-IRES-LIF. Hence, activation of the rPAP promoter could lead to LIF production and endogenous LIF receptor activation, which on its turn could again activate the rPAP promoter. The secreted LIF will stimulate and activate its own production in the same cell (autostimulation) as well as in surrounding cells (paracrine stimulation), resulting in a cascade of LIF production that ultimately exponentially amplifies the initial signal. Co-

transfection with the pXP2rPAP-luci construct or use of cells that have this plasmid stabile integrated allows for detection and quantification of the LIF cascade. To trigger the LIF cascade, we used an exogenous stimulus by LIF itself. The intensity of this initial trigger was varied by treating the cells with a LIF concentration gradient or with a fixed LIF concentration during different time periods (figure 3).

6x106 HEK293 cells were transfected in a petridish with 18 ug pUTrPAP-TK-IRES-LIF + 6 ug rPAP-luci DNA. In the negative control, pUTrPAP-TK-IRES-LIF was replaced by pUTrPAP-TK-IRES-IFNß. 24 hrs. after transfection, cells were trypsinized en transferred to a 24-well plate at a density of 3x105 cells/well. After another 24 hrs. incubation, a LIF concentration gradient ranging from 0 to 1 ng was added to the cells.

24 hrs. later, luciferase activity was determined in transfected cells by chemiluminescence. Briefly, cells were lysed in 300 pi lysis buffer (25 mM Tris, pH 7.8, with H3PO4 ; 2 mM EDTA; 2 mM DTT; 10% glycerol ; 1% Triton X-100) from which 100 , was transferred to a 96-well microtiterplate. 70 ut of luciferase substrate buffer (20 mM Tricine; 1.07mM (MgC03) 4Mg (OH) 2. 5H20 ; 2.67 mM MgS04. 7H20 ; 0.1 mM EDTA; 33.3 mM DTT; 270 uM Coenzyme A (lithium salt) ; 470 uM Luciferin (Duchefa); 530 uM ATP, final pH 7.8) was added to the 100 pI cell lysate and light emission was measured for 1 second in a Lumicount chemiluminescence counter (Packard). Results are shown in figure 3.

Several arguments point to a higher sensitivity for LIF of cells that express a LIF cascade: It is assumed that, during transient transfection, inducible promoters can be'leaky' because of the lack of repression factors (e. g. histones, repression factors...) that function only when the DNA is integrated in the genome. This may account for the high rPAP-luci induction seen in non-treated cells, transfected with rPAP-TK-IRES-LIF where small, background LIF levels may already trigger the LIF cascade.

After subtraction of the background, the amount of luciferase, induced by a certain LIF concentration is substantially higher in the pUTrPAP-TK-IRES-LIF transfected cells as compared to the negative control, suggesting that in the first cells, rPAP activation is increased exponential after triggering.

A clear increase in luciferase activity is observed at 3 and 300 pg/ml LIF in cells transfected with pUTrPAP-TK-IRES-LIF and pUTrPAP-TK-IRES-IFNß, respectively, indicating that the cells are about 100-fold more sensitive to LIF when expressing the LIF cascade construct.

Example 2 : transfection of stabile HEK rPAP-luci cells and treatment of the cells with a LIF concentration qradient.

In a second experimental setup, 6x106 HEK293 cells, stably transfected with the pXP2rPAP-luci construct (HEK rPAP-luci), were transfected with 25 iug pUTrPAP-TK- IRES-LIF or 25 pg of rPAP-TK-IRES-IFNß (negative control). The cells were further treated exactly the same way as described in example 1. Results are shown in figure 4.

Results show lower background levels of luciferase activity in the pUTrPAP-TK-IRES- LIF transfected cells, not treated with LIF, as compared to example 1. The stabile integration of rPAP-luci probably implies a lower copy number of this plasmid in the HEK rPAP-luci cells as compared to transient transfection. Revealing activation of the LIF cascade may therefore be less clear in cells containing a lower amount of rPAP- luci replicons. However, as seen in example 1, and suggesting a rPAP-LIF loop, the degree of rPAP activation induced by LIF is much higher in the pUTrPAP-TK-IRES-LIF transfected cells as compared to the negative control.

Example 3 : co-transfection with pXP2rPAP-luci and treatment of cells with a fixed LIF concentration during different time periods 6x106 HEK293 cells were transfected with 18 ug pUTrPAP-TK-IRES-LIF + 6 Eug rPAP- luci DNA. In the negative control, pUTrPAP-TK-IRES-LIF was replaced by pUTrPAP- TK-IRES-IFNß. 24 hrs. after transfection, cells were trypsinized en transferred to a 24- well plate at a density of 3x105 cells/well. After another 24 hrs. incubation, cells were <BR> <BR> stimulated with 1 ng/ml LIF during varying time periods (5 min. to 8 hrs. ) after which the cells were washed twice with medium without LIF. 24hrs. after the onset of stimulation, luciferase activity was determined by chemiluminescence as described above. Results are shown in figure 5.

Again, a high background was observed in the cells transfected with the pUTrPAP-TK- IRES-LIF loop, probably as a result of aspecific LIF production during transient transfection (see exp. 1). Despite this background, a difference in rPAP stimulation between LIF stimulated and non-stimulated cells was seen after 4 and 8 hrs. treatment with LIF. Shorter LIF incubation times did not result in such an effect. Moreover, no such effect was detectable in cells, transfected with the pUTrPAP-TK-IRES-IFNß construct, indicating that shorter LIF stimuli can be detected when cells contain the rPAP-LIF loop.